Masking background fluorescence and luminescence in optical analysis of biomedical assays

ABSTRACT

In a process for the quantitative optical analysis of fluorescently labelled biological cells  5,  a cell layer on a transparent support at the bottom  2  of a reaction vessel  1  is in contact with a solution  3  containing the fluorescent dye  4.  The sensitivity of analytical detection can be considerably improved if to the fluorescent dye  4  already present in addition a masking dye  9,  which absorbs the excitation light  6  for the fluorescent dye  4  and/or its emission light  7,  is added to the solution  3  and/or if a separating layer  10  permeable to the solution and absorbing and/or reflecting the excitation light  6  or the emission light  7  is applied to the cell layer at the bottom  2.  This process can also be used for improving the sensitivity in the quantitative optical analysis of a luminescent biological cell layer. The separating layer  10  must in this case be composed such that it has a high power of reflection for the luminescent light  11 . Analogously, these process principles can also be used in receptor studies for the masking of the interfering background radiation in the quantitative optical analysis of fluorescently or luminescently labelled reaction components. In this case, a receptor layer  12  at the bottom  2  of a reaction vessel  1  is in contact with a solution (supernatant  3 ) in which a fluorescent or luminescent ligand  13  is dissolved. The sensitivity and accuracy of the analytical detection can be considerably improved here if a masking dye  9  which absorbs the excitation light  6  for the fluorescent dye and/or its emission light or (in the case of luminescent ligands) the luminescent light is added to the supernatant  3.  Instead of the masking dye in the solution  3  or optionally as an additional measure, a separating layer  10  permeable to the solution  3  and absorbing and/or reflecting the excitation light  6  and/or the emission light or the luminescent light can be applied to the cell or receptor layer  12  at the bottom  2.

[0001] The invention originates from a process for the quantitativeoptical analysis of fluorescently labelled biological cells which are incontact with a fluorescent dye solution or of luminescent cells whichare applied to a transparent support at the bottom of a reaction vesselin the form of a coherent cell layer, or alternatively of fluorescentlyor luminescently labelled reaction components in a solution in which afluorescent or luminescent ligand is dissolved, the solution being incontact with a receptor layer, which is specific for this ligand andsituated on the transparent support at the bottom of the reactionvessel, whose fluorescent or luminescent radiation, which ischaracteristic of the receptor-ligand binding, is detected and analysedthrough the transparent bottom.

[0002] A problem in fluorescence measurement in biomedical assays isoften that the fluorescence changes correlated with the biological cellaction are small compared with the non-specific background fluorescence.As a result, the resolving power is greatly restricted. Conventionalcommercial measuring systems (fluorescence readers, Dynatech or SLT),cannot solve the problem, because owing to their optical measuringarrangement (excitation from ‘above’ through the fluorescent liquidcolumn of the supernatant) the signal can barely be detected incomparison with the background. Apparatuses of newer construction(Labsystems), which illuminate the cells from the back through thetransparent support of the reaction vessel, do have the advantage thaton entry of the excitation light the cells are excited to fluorescence.Since the excitation light, however, enters further into thesupernatant, which is also fluorescent, the fact that the non-specificbackground signal adulterates the cell signal cannot be avoided. Evenvery complicated measuring systems (NovelTech, FLIPR: FluorescenceImaging Plate Reader) are only able to decrease this backgroundfluorescence using a special laser illumination geometry (excitationbelow about 45°). The reason for the failure of all problem-solvingexperiments on the measuring geometry is the fact that the actual causeof the background fluorescence cannot be decisively influenced hereby.

[0003] In the receptor binding studies carried out until now usingfluorescently or luminescently labelled ligands, the labelled andunbound fraction in each case must be removed by processes like washing.Many coatings, however, are sensitive to these washing steps. Moreover,the removal of the unbound ligand is associated with a considerableoutlay. The direct measurement of the receptor-ligand association ordissociation is not possible in this process.

[0004] The invention is based on the object of improving the sensitivityof the optical analysis of fluorescently labelled or luminescent cellsin a cellular assay in order to be able to measure, for example,membrane potential changes which are as low as possible on the basis offluorescence changes of potential-sensitive dyes. In this case, thesensitivity of the measuring system should be so high that potentialchanges of below 5 mV can be detected at least qualitatively. In thecase of luminescent cells, an increase in the detection of theluminescence signal should be achieved. Moreover, the method should besuitable for screening with a high sample throughput.

[0005] The invention is furthermore based on the object of simplifyingreceptor binding studies based on fluorescently or luminescentlylabelled ligands or receptors and making possible continuous measurementof the receptor binding interaction (kinetics). Owing to the reductionin the process steps necessary, this method should be particularlysuitable for screening with a high throughput and for diagnosticapplications.

[0006] It was only possible to achieve the required high resolution withlow membrane potential changes after it was possible to eliminate thecause of the interfering overlapping of the non-specific backgroundfluorescence and the specific fluoresence of the cells. The processaccording to the invention developed for this purpose is based on thefundamentally new idea of masking the excitation energy and thefluorescence not originating from the biological object. To do this, inaddition to the fluorescent dye, a further dye is added which completelyabsorbs the excitation light of the fluorescent dye and/or its emissionlight without affecting the fluorescence of the cells. By means of thisabsorption, the non-specific background signal is masked and the usefulcell signal can be detected with a resolution which was previously notpossible.

[0007] An alternative solution which is within the scope of theinvention is that a separating layer, which is permeable to the solutionand which absorbs and/or reflects the excitation light for thefluorescent dye and/or its emission light without adversely affectingthe cell properties, is applied to the cell layer. At the same time, thethickness of the separating layer is selected such that fluorescence isno longer detectable in the dissolving mixture with the fluorescent dyebut without the cells.

[0008] A further variant of the invention is that the method of theseparating layer according to the invention is also used for increasingthe sensitivity in the quantitative optical analysis of luminescent(luminous) biological cells which are applied to a transparent supportin the form of a coherent cell layer. For this purpose, the opticalproperties of the separating layer permeable to the solution areselected such that it reflects the luminescent light as strongly aspossible without adversely affecting the cell properties. In thismanner, it is possible to increase the luminescence intensity and thusthe measured effect considerably.

[0009] The process according to the invention can be used in acompletely analogous manner for the quantitative optical analysis offluorescently or luminescently labelled reaction components in areaction vessel filled with a solution, the fluorescent or luminescentligand being present in dissolved form and the solution being in contactwith a receptor layer which is specific for this ligand, applied to atransparent support at the bottom of the reaction vessel or depositedthereon, whose fluorescent or luminescent radiation, which ischaracteristic for receptor-ligand binding, is detected and analysedthrough the transparent bottom. In this case, the solution according tothe invention of the object described above is based on the fact thatthe free ligand which is in the supernatant, i.e. in solution, and itsnon-specific fluorescence or luminescence is masked by an additional dyeand/or by a diffusely absorbing or reflecting separating layer and thusthe cause of the interfering overlapping of the non-specific backgroundfluorescence and the specific fluorescence of the ligand in the solutionis eliminated. Since the non-bound ligand is masked in this manner, themeasured fluorescence or luminescence is a direct measure of theligand-receptor interaction. It can be measured directly in this processwith time resolution.

[0010] In receptor studies, in analogy to the process described above,the invention thus relates to a process variant in which a masking dyeis added to the solution and/or a separating layer permeable to thesolution is applied to the receptor layer, the optical properties of themasking dye and/or of the separating layer being selected such that theexcitation light for the fluorescent dye of the ligand present in thesolution and/or its emission light or its luminescent light is absorbedby the solution or the separating layer or reflected at the separatinglayer. In this case, the thickness of the separating layer is selectedsuch that fluorescence is no longer detectable in the dissolving mixturewith the fluorescent dye, but without the receptor layer.

[0011] The separating layer preferably consists of polymeric latex beads(e.g. polystyrene, polyurethane, butadiene, acrylonitrile). The latexbeads can also be dyed with a masking dye, which in this case must havean adequately high polymer dyeing capacity.

[0012] In the first-mentioned process, the masking dye should be as welldistributed as possible in the solution which also contains thefluorescent dye in dissolved form. Since, as a rule, the solvent iswater, a masking dye is expediently employed which possesses good watersolubility (>2 g/ml) and has no cytotoxic side effects.

[0013] According to a further development of the invention, after thereplacement of the supernatant containing a fluorescent dye by afluorescent dye-free solution, a further masking dye is added whichsuppresses a non-specific fluorescence on the reaction vessel wall.

[0014] The following advantages are achieved using the invention:

[0015] The new process described is not tied to a certain measuringsystem, but can be used, because it is not a specifically technicalsolution, by many commercially available apparatuses. These includevirtually all fluorescence readers which can illuminate and also measuretransparent reaction vessels, e.g. microtitre plates, from the bottom.With a very low outlay (minimal additional costs only for the specialabsorption dyes), it is possible for the first time by this means toadvance in a resolving area, e.g. in the measurement of potentialchanges in cell membranes by measurement of the change in fluorescenceof potential-sensitive fluorescent dyes, which was unachieved until now.For the first time it is possible even in the case of very low changesto carry out a direct comparison of the results from various reactionvessels (e.g. various wells in a microtitre plate), such that thecomplicated procedure of the determination of the relative change in areaction vessel can be dispensed with. As a result the number ofmeasurements to be determined, e.g. for kinetic measurements, decreases.The outlay in terms of time for a measuring programme is markedlyreduced and the possibility created of obtaining identical results by asimple individual measurement (e.g. end point determination) with theuse of reference to a separate control batch. The uniformity of thebiological batch required in this case (e.g. homogeneous cell layer) isgenerally afforded, for example, for microtitre plates.

[0016] Surprisingly, the use of various water-soluble dyes and alsotheir mixtures in the very different cells tested showed no negativeeffect on the physiology of the cells (e.g. reaction of the cells incomparison with electrophysiological measurements such as whole-cellpatch-clamp, or effects of the pharmaceuticals investigated). The use ofundissolved dye pigments or inorganic finely divided particles was alsosurprisingly well tolerated by the biological objects.

[0017] As a result of the simple process described for masking thebackground fluorescence in quantitative fluorescence measurement inbiomedical assays, connected with an increase in the sensitivity, e.g.when using potential-sensitive fluorescent dyes, and the adaptability ofthis process, e.g. to microtitre plates as reaction vessels, the use ofsuch measuring techniques will significantly simplify high-throughputscreening, especially as no increased technical expenditure is necessaryfor the realization of the advantages outlined, but existing commercialmeasuring apparatuses are sufficient for this purpose. Inreceptor-ligand studies, the advantage essential to the invention isthat, on account of the masking of the non-specific fluorescence orluminescence, it is no longer necessary to remove the unbound fractionof the ligands. As a result, the test procedures are considerablysimplified, damage to and destruction of the sensitive coatings or ofthe biological objects such as, for example, cells are avoided and thesensitivity and thus also the accuracy of the measurement are improved.As a result of the use of microparticles, the utilizable surface areafor the coating of fluorescently or luminescently labelled ligands canbe significantly increased. By means of suitable measures, e.g.relatively high specific density or the use of magnetizable particles,the settlement and concentration of the microparticles on thetransparent support can be achieved. In this case too, the fluorescenceor luminescence of the unbound ligands in the supernatant is effectivelysuppressed by the masking.

[0018] Since the interaction between the ligands and the receptor mustnot be interrupted by the removal of the unbound fraction, a continuousmeasurement of the interaction between ligand and receptor (kinetics)can be carried out in this manner even in an individual reaction batch.

[0019] The invention is explained in greater detail below with the aidof working examples and drawings, wherein

[0020]FIG. 1 shows a reaction vessel for a fluorescence assay accordingto the prior art

[0021]FIG. 2 shows the suppression of the background fluorescence in afluorescence assay with a masking dye in the supernatant

[0022]FIG. 3 shows the spectral excitation and emission for a dispersiondye and the spectral absorption of the masking dye

[0023]FIG. 4 shows the site-dependent cell fluorescence without maskingdye

[0024]FIG. 5 shows the site-dependent cell fluorescence with masking dye

[0025]FIG. 6 shows the suppresion of the background fluorescence in afluorescence assay with the aid of a separating layer

[0026]FIG. 7 shows the amplification of the luminescence byback-reflection from a separating layer

[0027]FIG. 8 shows the wall fluorescence in a fluorescence assayaccording to the prior art

[0028]FIG. 9 shows the suppression of the wall fluorescence in afluorescence assay with the aid of a masking dye and

[0029]FIG. 10 shows the suppression of the background fluorescence orluminescence in a fluorescence or luminescence assay for theinvestigation of receptor-ligand binding with the aid of a masking dyein the supernatant

[0030]FIG. 1 shows a reaction vessel 1 for a fluorescence assay with atransparent bottom 2. A fluorescent dye solution 3, in which thefluorescent dye molecules 4 are indicated schematically, is found in thereaction vessel 1. The solution 3 is also designated as the supernatant.The biological cells to be investigated are arranged on a transparentsupport on the transparent bottom 2. Light (excitation light) 6 is shonethrough the bottom 2 in order to excite the cells 5 to fluorescence. Abackground fluorescence radiation 8, which originates from the likewiseexcited fluorescent dye molecules 4 in the supernatant 3, overlaps thefluorescent light 7 emitted by the cells 5. Only the fluorescent light7, however, is decisive for the bioanalytical investigation and analysisof the cells 5. Since, however, in all known fluorescence analysisapparatuses the background fluorescence 8 is additionally determined,small fluorescence differences of the cells 5 are lost in the strongbackground fluorescence 8, which leads to a marked sensitivity loss.

[0031] This disadvantage can be avoided by the procedure according tothe invention of FIG. 2 by suppressing the background fluorescence by amasking dye in the supernatant 3. The background fluorescence 8 presentin FIG. I is completely absorbed in the supernatant according to FIG. 2.The masking dye (schematically designated by 9) added to the supernatant3 can either be present in dissolved form or in finely divided dispersephase (colour-pigmented systems). Preferably, however, soluble dyes areemployed, because in this case the addition can be carried outparticularly simply with the aid of a pipette and because, in contrastto a pigment system, the physical effects of particle size distributionand of sedimentation processes and layer thickness inhomogeneities donot have to be taken into account.

[0032] The following demands are made on the properties of a dye of thistype:

[0033] when using a soluble absorption dye, good water solubility foruse in biological assays

[0034] no membrane permeability of the dye in order to avoid staining ofthe cells

[0035] high specific absorption in the excitation and/or emissionwavelength range of the fluorescent dye

[0036] no toxic side effects (avoidance of cell damage)

[0037] A solubility of >2 mg/ml is regarded as good water solubility.The cell toxicity can be determined with the aid of known testprocedures (e.g. cytotoxicity test). FIG. 3 shows the optical (spectral)properties of a fluorescent dye and of a masking dye in a graph. Curve Ashows the spectral distribution of the excitation light, curve B thespectral distribution of the emitted fluorescent light for thecommercially available dispersion fluorescent dyebis(1,3-dibutylbarbituric acid)trimethaneoxonol (Dibac₄(3)) and curve Cthe spectral transmission (absorption spectrum) of the masking dye used(Brilliant Black BN, C.I. 28440, Food Black 1, e.g. Sigma B-8384). It isrecognized that the masking dye is almost completely absorbed in thewavelength range of the excitation and emission of the fluorescent dye.

[0038] Contrast enhancement or increase in sensitivity can be evenbetter understood with the aid of FIGS. 4 and 5. To demonstrate theaction of the masking dye on the non-specific background fluorescence,two video recordings of the same image section were made before andafter adding 100 mg/ml of the soluble masking dye Brilliant Black in thepresence of the potential-sensitive fluorescent dye Dibac₄(3) (5 mM).Both times, the same video line was assessed by image analysis and thetwo fluorescence intensity profiles shown over the identical sections inthe reaction vessel. The region Z in this case corresponds to the regionin which the cell layer, i.e. the biological sample, is found, whileright of this in the zone Ü, to the greatest part, the fluorescencesignal originating from the supernatant is measured. The measuring rangeof the recording system (8 bit) is between 0 (black) and 255 (white).For the unmasked recording, a contrast ratio of about 1:3.6 results(intensity ratio of the darkest and lightest image portions) and in thecase of the masked recording a contrast ratio of about 1:14.4. Thiscorresponds to a contrast increase by a factor of 4.

[0039] According to FIG. 6, an alternative possibility of improving theratio of useful to background signal is covering of the cell layer witha finely divided optical separating layer 10. The separating layer 10expediently consists of a finely divided inorganic white pigment, suchas, for example TiO₂ or Al₂O₃. By means of this, not only the backgroundfluorescence radiation from the supernatant 3 is screened off, but alsothe measurable quantity of cell fluorescence is increased by reflectionfrom the inorganic particles.

[0040] Alternatively, the separating layer can consist of polymericlatex beads having a diameter preferably in the range from 200 nm to 5mm. Suitable polymers are, for example, polystyrene, polyurethane,butadiene, acrylonitrile. The latex beads can also be dyed with asuitable masking dye to which the same criteria apply as for theabsorption dye added to the solution (see above). A suitable class ofdye is, for example, ®Resoline.

[0041] In luminescence assays (luminous cells), the requirementsfundamentally consist of detecting the specific very low light intensityof a biological cell with high sensitivity.

[0042] By applying a reflecting separating layer 10 according to FIG. 7,it is possible, analogously to the method for suppression of thebackground fluorescence (according to FIG. 6), to increase theluminescence signal of the biological cells. In this connection,radiated fractions 11 of the undirected luminescent light are reflectedin the direction of the detector and thus increase the specificmeasuring signal.

[0043] In a large number of other fluorescent test procedures onbiological cells, it is possible, in contrast to dispersion dyes, toremove the fluorescent dye from the supernatant after staining of thecells by solution exchange. The fluorescent dye FURA2-AM is cleaved, forexample, into the free dye after penetrating into the cell and in thiscase loses its cell membrane permeability. As a result, a concentrationof the impermeable fluorescent dye in the cell occurs. In this case, thefluorescent supernatant 3 can be replaced by a fluorescent dye-freesolution 3 a without changing the specific cell fluorescence. Thenon-specific background fluorescence of the supernatant is removed inthis manner. FURA2-AM, however, stains reaction vessels persistently(wall fluorescence) and thus produces another non-specific fluorescencesignal which is comparable with the background fluorescence ofdispersion dyes. This situation is shown in FIG. 8. In this case, thebackground fluorescence radiation 8 is attributed to the fluorescent dyemolecules 4 adhering to the vessel walls. By incorporation of maskingdyes in the fluorescent dye-free supernatant 3 a, this non-specificfluorescence signal can also be completely suppressed.

[0044] In FIG. 10, a working example analogous to FIG. 2 is additionallyshown in which the biological layer applied to a transparent support atthe bottom 2 of the reaction vessel 1 consists of receptors 12 whichenter into a specific bond with the fluorescently or luminescentlylabelled ligand 13 present in the supernatant (solution) 3. The boundligands are designated here by 14. In the case of the unlabelledsolution, the primary light 6 shone through the bottom 2 excites thefluorescently labelled ligands 13 and 14 to fluorescence. In the case ofluminescently labelled ligands, the primary light 6 is inapplicable.Analogously to the implementation according to FIG. 2, a masking dyewhich takes care that the fluorescent or luminescent radiation emittedfrom the unbound ligands 13 is completely absorbed in the solution is inturn added to the solution 3. The fluorescent or luminescent radiation15 determined at the bottom 2, i.e. the measured effect, is thereforevery predominantly attributed to the ligands 14 bound to the receptors12 and is not adulterated by the background radiation of the unboundligands 13 in the solution 3. The measuring signal is therefore a directmeasure of the strength of the ligand-receptor binding. In this case,the layer thickness of the receptor layer is in the nm range, while thedimensions of the supernatant situated above it are in the order ofmagnitude of several mm.

[0045] According to FIGS. 6 and 7, the separating layer 10 permeable tothe solution can be employed in the investigation of ligand-receptorbinding in an entirely analogous manner to the masking or suppression ofthe background fluorescence or luminescence. In this case, theinterfering background fluorescence or luminescence is screened off bythe separating layer 10 and in the case of luminescent ligands radiatedfractions 11 of the luminescent light originating from bound ligands arereflected in the direction towards the reflector and thus the usefulsignal is increased.

[0046] The non-fluorescent or luminescent reaction component to beassessed with respect to its binding strength in classicalpharmacological receptor binding studies was not shown here for reasonsof clarity.

1. Process for the quantitative optical analysis of fluorescentlylabelled biological cells (5) which are applied to a transparent supportat the bottom (2) of a reaction vessel (1) in the form of a coherentcell layer and are in contact with a solution (3) containing thefluorescent dye (4), or of luminescent, biological cells in the form ofa coherent cell layer situated on the transparent support, characterizedin that the fluorescent dye (4) already present in addition to a maskingdye (9) which absorbs the excitation light (6) for the fluorescent dye(4) and/or its emission light (7) is added to the solution (3) and/or inthat a separating layer (10) which is permeable to the solution andwhich absorbs and/or reflects the excitation light (6) for thefluorescent dye (4) and/or its emission light (7) or, in the case of theluminescent cell layer, reflects the luminescent light, is applied tothe cell layer.
 2. Process for the quantitative optical analysis offluorescently or luminescently labelled reaction components in areaction vessel (1) filled with a solution (3) in which a fluorescent orluminescent ligand (13) is dissolved and the solution (3) is in contactwith a receptor layer (12), which is specific for this ligand (13) andis applied to a transparent support at the bottom (2) of the reactionvessel (1) or deposited thereon, whose fluorescent or luminescentradiation (7, 15), which is characteristic of the receptor-ligandbinding, is detected and analysed through the transparent bottom (2),characterized in that a masking dye (9) is added to the solution (3)and/or a separating layer (10) permeable to the solution (3) is appliedto the receptor layer (12), the optical properties of the masking dye(9) and/or of the separating layer (10) being selected such that theexcitation light (6) for the fluorescent dye (4) of the ligand (13)present in the solution (3) and/or its fluorescent light (8) or itsluminescent light is absorbed by the solution (3) or the separatinglayer (10) or reflected at the separating layer (10).
 3. Processaccording to claim 1 or 2, characterized in that the separating layer(10) used is a layer of polymeric latex beads.
 4. Process according toclaim 3, characterized in that the polymeric latex beads are dyed with amasking dye.
 5. Process according to claim 1-2, characterized in that amasking dye is used which possesses good water solubility and has nocytotoxic side effects.
 6. Process according to claim 1-2, characterizedin that in the case of a replacement of the supernatant (3) containing afluorescent dye (4) by a fluorescent dye-free solution (3 a) a maskingdye is added which suppresses the non-specific fluorescence emitted fromthe stained reaction vessel wall.